Generation of scRNA-seqand paired TCR-seq data on CD8+TILs from MC38 tumors in inducible PD-1del.mice. (A) Schematic of the experimental design for the scRNA-seq experiments. For Experiment (Exp) 1, roughly equal numbers of PD-1Hi and PD-1Lo CD8+ TILs were sorted from UBC-CreERT2+ PD-1f/f mice (referred to as inducible PD-1del. mice) or UBC-CreERT2− PD-1f/f mice (referred to as WT control mice) on day 17 (following administration of tamoxifen from days 7 to 11) and loaded onto the 10X Chromium Controller. Two mice were pooled for each genotype. For Experiment 2, total CD8+ TILs were sorted from UBC-CreERT2+ PD-1f/f mice or UBC-CreERT2− PD-1f/f mice on day 16 (following administration of tamoxifen from days 7 to 11) and loaded onto the 10X Chromium Controller, and PD-1 protein status was determined computationally by detection of PD-1 using CITE-seq. One mouse per genotype was run per channel, and two channels (so two mice) were run per genotype. Schematic was created with https://BioRender.com. (B) Flow cytometry contour plots showing the frequency of PD-1Hi and PD-1Lo CD8+ TILs in each of the samples that were loaded on the 10X Chromium Controller. An aliquot of each sample was subjected to fluorescent-based PD-1 protein analysis using flow cytometry to confirm expected frequencies of PD-1Hi and PD-1Lo. (C) Sample details from 10X runs. (D) Representative histograms of distribution of counts for PD-1 CITE-seq (Feature Barcode detection). Shown is one mouse of each genotype from Experiment 2 (Mouse 3 = WT control, Mouse 5 = inducible PD-1del.), representative of both mice in each genotype in this experiment. A threshold was set (shown in the dotted line) to classify cells as PD-1Hi or PD-1Lo, with anything above that threshold was set as PD-1Hi, and anything below that threshold was PD-1Lo. (E) Distribution of each sample based on genotype (WT versus inducible PD-1del.) and PD-1 protein status in Experiment 1 (Mouse 1 and Mouse 2). (F) Distribution of each sample based on genotype (WT versus inducible PD-1del.) and PD-1 protein status in Experiment 2 (Mouse 3–6). Plots split based on whether cells were classified as PD-1 protein high or low based on the threshold set in Fig. S1 D.
Figure S1.

Generation of scRNA-seq and paired TCR-seq data on CD8 + TILs from MC38 tumors in inducible PD-1 del. mice. (A) Schematic of the experimental design for the scRNA-seq experiments. For Experiment (Exp) 1, roughly equal numbers of PD-1Hi and PD-1Lo CD8+ TILs were sorted from UBC-CreERT2+ PD-1f/f mice (referred to as inducible PD-1del. mice) or UBC-CreERT2− PD-1f/f mice (referred to as WT control mice) on day 17 (following administration of tamoxifen from days 7 to 11) and loaded onto the 10X Chromium Controller. Two mice were pooled for each genotype. For Experiment 2, total CD8+ TILs were sorted from UBC-CreERT2+ PD-1f/f mice or UBC-CreERT2− PD-1f/f mice on day 16 (following administration of tamoxifen from days 7 to 11) and loaded onto the 10X Chromium Controller, and PD-1 protein status was determined computationally by detection of PD-1 using CITE-seq. One mouse per genotype was run per channel, and two channels (so two mice) were run per genotype. Schematic was created with https://BioRender.com. (B) Flow cytometry contour plots showing the frequency of PD-1Hi and PD-1Lo CD8+ TILs in each of the samples that were loaded on the 10X Chromium Controller. An aliquot of each sample was subjected to fluorescent-based PD-1 protein analysis using flow cytometry to confirm expected frequencies of PD-1Hi and PD-1Lo. (C) Sample details from 10X runs. (D) Representative histograms of distribution of counts for PD-1 CITE-seq (Feature Barcode detection). Shown is one mouse of each genotype from Experiment 2 (Mouse 3 = WT control, Mouse 5 = inducible PD-1del.), representative of both mice in each genotype in this experiment. A threshold was set (shown in the dotted line) to classify cells as PD-1Hi or PD-1Lo, with anything above that threshold was set as PD-1Hi, and anything below that threshold was PD-1Lo. (E) Distribution of each sample based on genotype (WT versus inducible PD-1del.) and PD-1 protein status in Experiment 1 (Mouse 1 and Mouse 2). (F) Distribution of each sample based on genotype (WT versus inducible PD-1del.) and PD-1 protein status in Experiment 2 (Mouse 3–6). Plots split based on whether cells were classified as PD-1 protein high or low based on the threshold set in Fig. S1 D.

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